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Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler
Peizhi Zhu1, Jean-Pierre Clamme, Ashok A Deniz
1Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Biophysical Journal
|October 4, 2005
Summary
This study shows that TEMPO-5-TAMRA fluorescence quenching can measure short distances within biomolecules. This method accurately quantifies intramolecular distances between 10-30 Angstroms using ensemble and single-molecule experiments.
Area of Science:
- Biophysics
- Chemical Biology
- Molecular Biophysics
Background:
- Measuring short intramolecular distances is crucial for understanding biomolecular function.
- Fluorescence quenching assays offer potential for distance measurements but require precise calibration.
Purpose of the Study:
- To investigate the use of intramolecular fluorescence quenching by TEMPO for measuring short distances (10-30 Å) in DNA molecules.
- To validate the method using both ensemble and single-molecule techniques.
Main Methods:
- Synthesis of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the nitroxide radical TEMPO.
- Steady-state ensemble fluorescence and fluorescence correlation spectroscopy (FCS) measurements.
- Molecular mechanics modeling for distance calculations.
Main Results:
- An exponential decrease in quenching rate constant was observed with increasing dye-quencher distance.
- The TEMPO-5-TAMRA system demonstrated reliable distance measurements in the 10-30 Å range.
- Both ensemble and single-molecule experiments yielded consistent results.
Conclusions:
- TEMPO-5-TAMRA fluorescence quenching is a viable and promising method for determining short intramolecular distances.
- This technique can be applied to study distance changes within single biomolecules.
- The findings support the use of fluorescence quenching for nanoscale distance measurements in biophysical studies.